Chemico-Biological Interactions 259 (2016) 122e132 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint Design and synthesis of N-substituted-2-hydroxyiminoacetamides and interactions with cholinesterases Nikola Marakovic a, Anamarija Knezevic b, Vladimir Vinkovic b, Zrinka Kovarik a, * Goran Sinko a, a Institute for Medical Research and Occupational Health, Ksaverska cesta 2, HR-10 000 Zagreb, Croatia b RuCer Boskovic Institute, Bijenicka cesta 54, HR-10 000 Zagreb, Croatia article info abstract Article history: Within this study, we designed and synthesized four new oxime compounds of the N-substituted 2- Received 9 January 2016 hydroxyiminoacetamide structure and evaluated their interactions with acetylcholinesterase (AChE) Received in revised form and butyrylcholinesterase (BChE). Our aim was to explore the possibility of extending the dual-binding 20 April 2016 mode of interaction between the enzyme and the inhibitor to a so-called triple-binding mode of inter- Accepted 25 May 2016 action through the introduction of an additional binding moiety. N-substituted 2- Available online 26 May 2016 hydroxyiminoacetamide 1 was prepared via BOP catalyzed amidation of hydroxyiminoacetic acid with 3-azido-1-phenylpropylamine. An azide group enabled us to prepare more elaborate structures 2e4 by Keywords: e Oxime antidotes the copper-catalyzed azide-alkyne cycloaddition. The new compounds 1 4 differed in their presumed Azide-alkyne cycloaddition AChE peripheral site binding moiety, which ranged from an azide group to functionalized heterocycles. Organophosphorus compounds Molecular docking studies revealed that all three binding moieties are involved in the non-covalent Inhibition interactions with ChEs for all of the four compounds, albeit not always in the complete accordance Selectivity with the proposed hypothesis. All of the four compounds reversibly inhibited the ChEs with their in- hibition potency increasing in the same order for both enzymes (1 < 2 < 4 < 3). A higher preference for binding to BChE (KI from 0.30 mmol/L to 130 mmol/L) over AChE (KI from 50 mmol/L to 1200 mmol/L) was observed for all of the compounds. Compounds were screened for reactivation of cyclosarin-, sarin- and VX-inhibited AChE and BChE. © 2016 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Gly122, Ala204), an acyl-binding pocket (Phe288, Phe290) and a choline binding site (Trp86, Tyr337, Phe338) [4,5]. The two sub- Acetylcholinesterase (AChE; EC 3.1.1.7) is a key enzyme for the sites serve as the recognition sites for the ligands that bind to the regulation of cholinergic transmission in both the central and pe- AChE governing their mechanism of interaction. Depending on the ripheral nervous system that catalyzes the hydrolysis of the established interactions, ligands can be described as PAS-binding or neurotransmitter acetylcholine (ACh) [1]. The decline of hippo- the CAS-binding. Out of several anti-AD drugs (Fig. 1), galanth- campal and cortical levels of ACh is a characteristic of Alzheimer’s amine (half-maximal inhibitory concentration (IC50) of 2.01 mmol/L disease (AD), a disabling and fatal neurodegenerative disease in human AChE) [6,7] and huperzine A (IC50 of 0.082 mmol/L in manifested by memory loss and learning deficits [2]. Thus, today’s mouse AChE) [8,9] bind to the CAS, while propidium iodide (IC50 of drugs designed for the treatment of AD are reversible AChE in- 1.1 mmol/L in mouse AChE) [10] binds in the PAS region [11]. In- hibitors that block the enzyme active site leading to an increase of hibitors that bind to PAS and CAS simultaneously include both ACh levels and, in turn, to the alleviation of disease symptoms [3]. symmetrical (e.g. bistacrine, Kd of 250 nmol/L in fetal bovine serum The active site of AChE is a 20 Å deep gorge divided into two sub- AChE) [12,13] and non-symmetrical tacrine analogues (e.g. syn- sites; the peripheral anionic site (PAS) (Tyr72, Tyr124, Trp286) TZ2PA6, Kd of 0.41 nmol/L in mouse AChE) [14,15],aswellas located at the entrance of the gorge, and the catalytic site (CAS) donepezil (Kd in hAChE of 3.35 nmol/L (R-donepezil), 17.5 nmol/L located close to the bottom of the gorge. CAS is composed of the (S-donepezil)) an anti-AD drug that binds with a basic nitrogen in catalytic triad (Ser203, His447, Glu334), an oxyanion hole (Gly121, the CAS and an aromatic system in the PAS [16,17]. Crystal struc- tures of AChE-inhibitor complexes have shown that inhibitors * Corresponding author. usually interact with gorge residues via areneearene (p-p) E-mail address: [email protected] (G. Sinko). http://dx.doi.org/10.1016/j.cbi.2016.05.035 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved. N. Marakovic et al. / Chemico-Biological Interactions 259 (2016) 122e132 123 Fig. 1. Structures of cholinesterase inhibitors, and reactivators of organophosphorus nerve agent-inhibited cholinesterases. interactions. Trp86, which is essential for the interaction with the [8,17,23,24]. Several selective BChE inhibitors have also been trimethylammonium group of ACh, is considered crucial for the described, including bambuterol and ethopropazine (Fig. 1) [41e45]. stabilization of CAS-binding ligands through cation-p and p-p in- Using ChE active site differences for designing selective inhibitors teractions together with the Tyr337, and His447 of the catalytic could help develop improved AD drugs and reactivators of OP nerve triad [18e20]. On the other hand, PAS-binding ligands are stabilized agent-inhibited enzymes. through p-p interactions with Tyr72, Tyr124, and Trp286 [21e23]. In this study, we designed and synthesized four new compounds The acute toxicity of organophosphorus (OP) nerve agents (e.g. to probe the possibility of simultaneous non-covalent triple-bind- tabun, soman, sarin, VX) is due to their irreversible inhibition of ing between the inhibitors and the ChE. Our results could lead to AChE by covalently binding to the catalytic serine residue which the discovery of more selective ChE inhibitors as well as more results in the accumulation of ACh in synaptic clefts [24]. The activity effective reactivators of OP nerve agent-inhibited enzymes. Com- of AChE can be restored by treatment with a reactivator from the pounds were designed by modifying the structures of N- quaternary pyridinium oxime family, such as 2-PAM (KI for mAChE of substituted 2-hydroxyiminoacetamides, recently introduced non- € 150 mmol/L) [25], trimedoxime (KI for hAChE of 18 mmol/L) [26],Hlo- charged AChE oxime reactivators [46,47], through the introduc- 7(KI for hAChE of 24 mmol/L) [26],HI-6(KI for hAChE of 20 mmol/L) tion of a phenyl ring. It was expected that the phenyl ring would [27], obidoxime (Fig. 1), by cleaving the covalent bond between the help their stabilization through p-p interactions with active site catalytic serine residue and the nerve agent [28,29].HI-6isan aromatic amino acids and provide additional binding moiety apart example of a quaternary bis-pyridinium mono-oxime that binds to from PAS- moiety and the 2-hidroxyiminoacetamide group. The both the CAS and the PAS with its two positively charged hetero- working hypothesis was that the phenyl ring would interact with cyclic aromatic rings [30].Theefficacy of both AChE inhibitors and the choline binding site directing, together with PAS-binding reactivators currently used in medical treatment of AD or OP nerve moiety, the 2-hidroxyiminoacetamide group into a so-called third agent poisoning is limited because they do not cross the blood-brain binding site surrounding Ser203. Molecular modeling was used to barrier readily due to their permanent positive charge [31]. determine and visualize the binding modes of the new compounds Butyrylcholinesterase (BChE, E.C. 3.1.1.8) is related to AChE and it and their interactions with the enzymes. can also catalyze the hydrolysis of ACh; moreover, it serves as a co- regulator of cholinergic neurotransmission [32,33]. However, BChE 2. Materials and methods plays an important role in the pathogenesis of AD with its activity increased at the early stage of disease and involvement in the am- 2.1. Chemicals yloid b-peptide aggregation developing into senile plaque deposits [34,35]. The inhibition of BChE may thus be beneficial in the medical N-substituted 2-hydroxyiminoacetamides N-(3-azido-1- treatment of AD patients. AChE and BChE show a high resemblance phenylpropyl)-2-hydroxyiminoacetamide (1), N-(3-(4- with sequence homology of 65% [35,36]. However, their active sites cyclopentyl-1H-1,2,3-triazol-1-yl)-1-phenylpropyl)-2- display different amino acids composition and therefore the BChE hydroxyiminoacetamide (2), 2-hydroxy-imino-N-(3-(4-((2- active site is about 200 Å3 bigger [37,38],consequentlyallowingthe methyl-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol-1-yl)-1- BChE to bind and hydrolyze larger ligands and substrates than AChE phenylpropyl)acetamide (3), and 2-hydroxyimino-N-(3-(4-((2- [39]. Moreover, differences between AChE and BChE active site hydroxyiminomethyl)-1H-imidazol-1-yl)methyl)-1H-1,2,3-triazol- amino acid composition lead to AChE/BChE selectivity for many li- 1-yl)-1-phenylpropyl)acetamide (4) were synthesized. 1 was pre- gands and substrates [23,40]. Some of the selective AChE inhibitors pared via BOP catalyzed amidation of hydroxyiminoacetic acid with are BW284C51, huperzin A, and the aforementioned donepezil 3-azido-1-phenylpropylamine [48]. 2e4 were prepared by copper 124 N. Marakovic et al. / Chemico-Biological Interactions 259 (2016) 122e132 catalyzed azide-alkyne cycloaddition starting from 1 and a corre- (dissociation) constant of a complex formed at the catalytic site, aKI sponding alkyne: ethynylcyclopentane, 2-methyl-1-(prop-2-yn-1- is the Michaelis complexeoxime inhibition (dissociation) constant yl)-1H-imidazole, and 1-(prop-2-yn-1-yl)-1H-imidazole-2- of a complex formed at the peripheral site, Km is a dissociation carbaldehyde oxime, respectively [49,50].